Cytodiagnostic method using alstonine as a selective marker, and diagnostic kit containing marker

ABSTRACT

An alstonine composition is useful as a selective marker of tumor cells and/or of chromosomal aberrations, and for the detection thereof by measurement of fluorescence at about 375 nm. Thus, an alstonine preparation can be used as a diagnostic agent designed for selective detection of tumoral diseases, and in cytogenetics. The diagnostic agent has application in preoperative, peroperative and postoperative diagnoses.

The present invention relates to cytodiagnostic techniques. Moreparticularly, it relates to the detection of tumors, notably for theestablishment of a tumoral diagnosis, and to the detection ofchromosomal aberrations by means of alstonine, using a procedure ofselective detection by fluorescence.

In the present description, the term "cytodiagnostic" is understood toinclude both the diagnosis of tumoral diseases and cytogenetics.

Alstonine is an alkaloid extracted from Rauwolfia vomitoria, Rauwolfiaobscura and Vinca rosea, as well as from other Apocyanaceae. It belongsto the group of quaternary beta-carbolines. Methods of extraction ofthis alkaloid have been described by M. Beljanski and J. Bugiel inFrench Patent Publication FR-A-78 30663 and European Patent PublicationEP-A-0 059 817, whose content is incorporated herein by reference. Ithas also been shown that this alkaloid has a very vivid pale bluefluorescence, which on spectrophotometric analysis corresponds to twoabsorption maxima, at 252 and 308 nm, respectively, which arecharacteristic of alstonine and of its isomer serpentine.

It has also been shown, notably in the above-mentioned documents, thatalstonine has anti-cancerous properties. These properties are now wellsubstantiated on mouse YC8 lymphoma ascites cells; on Ehrlich "ascites"tumor cells in mice; on type KB, HELA, HEPII and L cancer cell cultures;and on cells originating from nephroblastoma, neuroblastoma or teratoma,all of which are human tumors, primary cultures of which alstonine wasfound to destroy completely within 24 to 48 hours. By way of comparison,in order to firmly establish the selectivity of action of alstonineagainst cancerous cells, the same alstonine dose (200 mcg per ml ofculture) was applied to primary cultures of monkey kidney cells or toVero or Circ-type cell lines; in these cases, it produced no cellulardestruction within 8 days. This proved the specific interaction ofalstonine with the DNA of tumor cells, whereas the DNA of normal cellswas not affected by this alkaloid.

However, it was still difficult to have good control of an antitumortreatment using such an alkaloid and/or to establish a reliablediagnosis of a patient's condition in the preoperative, peroperative andpost-operative stages. Hence, there was a need for a means for theevaluation of tumoral diseases in order to facilitate the diagnosis ofcancerous lesions and of metastases, and to improve their treatment.

SUMMARY OF THE INVENTION

It has now been unexpectedly found that alstonine itself, can be used toobtain a selective diagnosis of tumoral diseases both with regard totheir location and to provide an estimate of their size. It has alsobeen found that a similar alstonine composition is useful incytogenetics. Thus, in accordance with the invention, a method isprovided for the detection of tumor cells or of chromosomal aberrationsin a tissue sample, comprising treating the tissue sample with acomposition comprising alstonine or an isomer thereof, irradiating thetreated tissue sample with light having a wavelength effective to inducea fluorescent emission at about 446 nm from alstonine absorbed intotumor cells or cells having a chromosomal aberration; and detecting anyfluorescence from the tissue sample. This method can be used for both invivo and in vitro diagnoses.

The invention also relates to kits for performing cytodiagnosticevaluations on tissue samples in accordance with the method of theinvention. Such a kit includes

(a) a composition comprising alstonine or an isomer thereof at aconcentration effective to impart measurable fluorescence to tumor cellsor to cells having chromosomal aberrations, and

(b) an incubation support adapted for incubation of the tissue sample inthe composition.

The invention further relates to an article of manufacture comprising apackaging material and a cytodiagnostic agent contained within saidpackaging material, wherein said cytodiagnostic agent comprisesalstonine or an isomer thereof at a concentration effective to impart adetective level of fluorescence to tumor cells, and wherein saidpackaging material comprises a label which indicates that saidcytodiagnostic agent can be used for the detection of tumors andchromosomal aberrations. In one embodiment, the cytodiagnostic agent isprovided as a galenic preparation in the form of a tablet or capsule fororal administration.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described below in greater detail by reference to theappended figures, wherein

FIG. 1 shows the excitation and emission spectra of alstonine;

FIG. 2 shows the absorption spectrum of alstonine;

FIG. 3A shows the variation of the intensity of fluorescence as afunction of the alstonine concentration;

FIGS. 3B and 3C are plots showing that this variation is linear in thealstonine concentration range of from 10⁻⁶ to 10⁻⁵ ;

FIGS. 4A to 4D show the results of the analysis of certainphysical-chemical parameters of alstonine incubated in accordance withthe invention in the two cell populations SK0 and ST3, carried out bymeans of a fluorescence imaging analyzer;

FIGS. 5A to 5C are diagrams showing the results of a Sephadex gelchromatography, using 10 mM tris-HCl buffer, pH 7.5, as eluent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention takes advantage of the facts that alstonine andits isomer serpentine accumulate in tumor cells, or cells havingchromosomal aberrations, but not in normal cells, and can be detected byfluorescence measurements. Thus, tumor cells or cells having chromosomalvariations can be distinguished from normal cells for cytodiagnosticpurposes by treatment with alstonine and observation of the treatedcells for fluorescence at a wavelength characteristic of alstonine.

As shown in FIG. 1, which shows the excitation and emission spectra of a5×10⁻⁶ M solution, alstonine excited at the three wavelengths of 260,310 and 375 nm produces fluorescence in the visible range (446 nm) withquantum yields which differ from one maximum to the other. The strongestemission corresponds to the excitation maximum at 310 nm. The UVabsorption spectrum showed three absorption bands at about 260, 310 and320 nm, respectively. (FIG. 2)

The quantum yield, which measures the ratio of emitted intensity toexcitation intensity, makes it possible to classify the fluorescenceexcitation maxima according to their efficiency. The maximum efficiencyis obtained at an excitation at 310 nm; the yield is lower but stillvery similar at 375 nm; by contrast, it is very low at an excitationwavelength of 260 nm. Because of the usual light absorption propertiesshown by optical fibers, an excitation wavelength of 260 nm would beunsuited for use with a conventional endoscope. Similarly, using anexcitation wavelength of 310 nm is generally not compatible with mostfluorescence instrumentation, because the glasses employed influorescence detection apparatus are transparent only above 360 nm.Hence, it is the existence of an efficient excitation wavelength at 375nm that makes the present invention involving detection of alstonine byfluorescence feasible with existing equipment.

Furthermore, it was established that at constant excitation intensity alinear relationship exists between the intensity of fluorescence and thealstonine concentration. As indicated by the results shown in FIGS. 3Ato 3C, at constant excitation intensity, the intensity of fluorescenceof alstonine and the concentration of this alkaloid are in a linearrelationship in an alstonine concentration range suitable for detectionby instruments conventionally used in spectrofluorometry. This was foundto be true particularly in an alstonine concentration range sufficientfor the detection of alstonine but low enough so as to minimize thetoxicity risks of the product with regard to healthy cells.

Studies carried out by chromatography on Sephadex gel columns (FIGS. 5Ato 5C) showed that there is no interaction between the double-strandedDNA or single-stranded DNA isolated from normal cells and alstonine.These results are in agreement with the publications on alstonine whichspecify that the latter accumulates only in tumor cells, due to itsinteraction with tumoral DNA [on this subject see, in particular, M.Beljanski and M. S. Beljanski, Exp. Cell. Biol., S. Karger AG, Basel,Pub. 50: 79-87 (1982), and M. Beljanski and M. S. Beljanski, Oncology,S. Karger AG, Basel, Pub. 43: 198-203 (1986)].

With regard to the alstonine accumulation studies conducted on live cellcultures of both normal cells (ST3) and tumor cells (SK0), they show(FIGS. 4A to 4D) that after incubation for about 20 minutes or more inthe presence of the product, the fluorescence studies carried out with aSamba 2000 imaging analyzer detect a major accumulation only in the SK0cells.

This accumulation of alstonine can be used in accordance with theinvention to detect tumor cells in a tissue sample in vitro. The tissuesample is incubated in a composition containing alstonine, e.g.,alstonine in aqueous solution at a concentration of about 0.2 to 5 mgper 20 ml of water, i.e. at a concentration of about 1 to 25% w/v. Theincubation is advantageously done over a period of about 20 to 40minutes at ambient temperature.

In in vitro applications, once this incubation stage has been completed,the tissue sample is rinsed with alcohol, preferably methanol, and areading is made by irradiating the incubated sample and observing anyfluorescence, for example with the fluorescence microscope.

For the preparation of a diagnostic kit usable mainly in in vitro tests,it is recommended to employ, as raw material, a purified saturatedaqueous solution of alstonine, and to prepare, at the time of use,dilutions of this same solution in pure water for analyses at usefulconcentrations, which, in practice, range between about 5 and 10% w/v.

For application of the method of the invention in vivo, it isrecommended to administer the alstonine in an appropriate galenicformulation, comprising suitable conventional pharmaceutical carriers orsupports and possible excipients, For example, a form such as tablets orgelatin capsules advantageously containing about 3 to 500 mg ofalstonine per unit dose is suitable. The administration is effected bythe oral route t.i.d. on the day before the invivo diagnosticexploration.

For clinical use of alstonine and its isomers in accordance with theinvention, notably in preoperative, peroperative and/or postoperativediagnosis and in the diagnosis of cutaneous diseases in confirmation ofother examinations, it is recommended to detect the fluorescence underWood's lamp excitation. This luminescence under Wood's lamp is utilizedeither by direct illumination or by transmission, notably by opticalfibers in the case of endoscopic explorations.

As a variant to the detection of tumor cells, an alstonine compositioncan also be advantageously used according to the invention incytogenetics, notably in investigations of an aberrant chromosomeidentification, through the detection, on the chromosomal level, ofbreaks, modifications and/or recombinations. In practice, thisidentification of chromosomal aberration, characteristic of a hereditarygenetic disease, is effected through a specific luminescence band of oneor more chromosomes involved in said chromosomal aberration and detectedby the technique of the present invention.

As an example, diagnostic studies based on an alstonine composition inaccordance with the invention and performed according to appropriatebiological test protocols, have provided cytogenetic confirmation of thecorrelations between the loss of one of the two sex chromosomes and theestablishment of solid tumors or malignant blood diseases:

In the case of female subjects, the karyotype then becomes 45×0 by lossof a chromosome X;

In the case of male subjects, the karyotype then becomes 45×0, by lossof a chromosome Y.

The mechanism of cytogenetic action controlling these events may thus beevaluated qualitatively and quantitatively by differential colorationand lymphocyte culture studies, which make it possible to demonstratethe bridges and breaks and the recombinations.

Although the above description and the following illustrative examplesmake reference to alstonine as a particular alkaloid, persons skilled inthe art will readily see that comparable results may be obtained if thisalkaloid is totally or partially replaced by any equivalent techniquefor the use under consideration, notably by its isomer serpentine.

EXAMPLES OF USE FOR A DIAGNOSIS BASED ON BIOLOGICAL SPECIMENS Example 1

Various biological specimens were impregnated with an aqueous solutionof alstonine containing about 200 micrograms of alstonine permilliliter; these specimens were incubated for 20-40 minutes, thensubjected to microscopic examination under a Wood's lamp microscope.

Treated and examined in this manner was a smear of uterine cervixspecimen on a glass slide, and the cancerous nature of the cellularelements was demonstrated by the luminescence observed under the Wood'slamp microscope.

Example 2

The procedure described in Example 1 was applied to a bone marrow smearon a glass slide.

The luminescence observed under the Wood's lamp microscope revealed thecancerous nature of the cellular elements.

Example 3

Proceeding as in Example 1 with a gastric specimen centrifuged and thenspread on a glass slide, the presence of tumor cells was demonstrated bythe luminescence of the smear on the slide, detected by the selectivemethod of the invention.

Example 4

Proceeding as in Example 1, with a vesical specimen spread on a glassslide, the presence of tumor cells was demonstrated by the luminescenceof the smear on the slide detected by the selective method of theinvention.

The means and method according to the present invention are also usefulfor in-vitro cytogenetic detection of chromosomal aberrations linkedwith a hereditary genetic disease, this detection being effected byidentification of a specific luminescence band of one or morechromosomes involved in breaks, modifications or recombinations.

EXAMPLES OF CLINICAL USE Example 5

On the day before the diagnostic exploration, one gelatin capsulecontaining 250 mg of alstonine, prepared as indicated above, was orallyadministered t.i.d. at more or less equal intervals during the day, topatients in whom ovarian or vesical tumors were to be investigated andevaluated. Endoscopic examination done on the day following theadministration of the capsules, with transmission through the endoscopeof UV excitation radiation generated by a Wood's lamp, made it possibleto confirm that the detection of neoplastic tumors was easy by thismethod. It was also established that the differential diagnosis withcancerous tumors was confirmed by biopsy and anatomopathologicexamination.

Example 6

According to the procedure indicated in Example 5, a diagnosis ofpancreatitis with development of a Weber-Christian type ofextrapancreatic vasculitis was made in patient Mr. A. G., showing atorpid course for 10 years. In this way it was significantly verifiedthat the consolidation did in fact occur after these ten years.

Example 7

According to the method indicated in Example 5, the diagnosis of aMerkel tumor was confirmed in a 74-year-old woman; this diagnosis wasalso confirmed by histological analyses of the excision of an APUD-typeneuro-endocrine carcinoma tumor.

Example 8

According to the procedure indicated in Example 5, a generalizedichtyosis was detected under UV in young S. L. aged 8 years; in this waythe presence of a thick keratosis without oligophrenia was noted. Onsubsequent repetition of the investigation according to the invention,the improvement resulting from the treatment was confirmed, as reflectedby clearing of the face, lower limbs and upper limbs, but withpersistence of the ichtyosis on the scalp, abdomen, chest and back.

Example 9

Using the procedure described in Example 5, a senile-type facialepitheliomatosis with development toward a right frontal basal cellformation was analyzed and located in a patient. On repeating theinvestigations of the present invention at suitable time intervals, theeffects of the treatment administered were followed until filling up ofthe basocellular formation and its cicatrization.

Example 10

The procedure described in Example 5 was used in patient Mr. C. Y.presenting a cutaneous lymphoma of the type of Sesary's disease. DuringUV exploration after administration of alstonine under the conditionsindicated in Example 1, this patient presented a lymphoid infiltrationof the face of the leontiasis type, as well as zones of dermoepidermitisand pseudoplaques of leukodysplasia of the face and lips.

Example 11

In patient Mr. D. F., subjected to the same procedure as described inExample 5, the marking action of alstonine under UV revealed theexistence of a recurrent fibrosarcoma after failure of its treatment ata time five years before this observation under UV. Monitoring of thetreatment with alstonine under UV showed disappearance of thecicatricial luminescence, which demonstrated the recovery of the normaltissue at the tumor site.

Example 12

Using the procedure described in Example 5, the existence of arectosigmoid adenocarcinoma was established and visualized by thefluorescence of alstonine in patient Mr. K., in whom this diagnosis wasconfirmed by the anatomopathologic study of the excision of a recurrentvillose lesion, showing, in particular, the presence of a Stage Iproliferative adenocarcinomatous focus.

Example 13

Using the procedure of Example 5 and after total gastrectomy in patientMr. A. J. due to the existence of a neoplastic mass at the lessercurvature of stomach and celiac lymph-node chain, the luminescence ofthe surgical specimen made it possible to establish the topography ofthe hyperluminescent zones, corresponding to the sites of neoplasticulcerations of the lesser vertical curvature.

Example 14

Proceeding as described in Example 5, patient Mrs. E. Y., whosubsequently underwent laparotomy after ultrasonic exploration, showed,thanks to the luminescence of alstonine after the laparotomy, theexistence of intensive luminescence over one ovary, and it was verifiedthat the patient in effect suffered from a metastatic proliferation of aprimary breast tumor.

Example 15

Proceeding as in Example 5, patient Mr. G. M. was treated by contactcobalt therapy for a spinocellular tumor of the ala nasi. Theluminescence of alstonine made it possible to demonstrate the recurrenceof dysplasia nasoorbital modifications on the opposite site (ricocheteffect).

Comparable trials carried out with other betacarbolines havingequivalent fluorescence properties, mainly with serpentine, have yieldedsimilar results and thus constitute techniques equivalent to alstoninewithin the framework of the present invention.

The means according to the invention are thus remarkably useful forcytodiagnosis, which comprises the evaluation of tumoral diseases andcytogenetics. In vivo, they make it possible, with a precision and easethat have never been attained so far, to locate a tumor in thepreoperative stage; in the peroperative stage, to make a study of thecancerous lesions and metastases thanks to the fluorescence; and in thepostoperative stage to verify, by fiber endoscopy, whether metastasesappear or progress after an anticancer treatment or chemotherapy usingtraditional methods.

I claim:
 1. A method for the detection of tumor cells or of chromosomalaberrations in a tissue sample, comprising treating the tissue samplewith a composition comprising alstonine or an isomer thereof,irradiating the treated tissue sample with light having a wavelengtheffective to induce a fluorescent emission having a wavelength of about446 nm from alstonine absorbed into tumor cells or cells having achromosomal aberration; and detecting any fluorescence from the tissuesample.
 2. A method according to claim 1, wherein the irradiating lighthas a wavelength of about 375 nm.
 3. A method according to claim 1,wherein the composition comprises alstonine in an aqueous solution at aconcentration of 1 to 25% by weight per volume.
 4. A method according toclaim 1, further comprising the step of diluting a purified saturatedaqueous solution of alstonine at the time of use, to form a compositionfor treating the tissue sample having an alstonine concentration ofbetween about 5 and 10% by weight per volume.
 5. A method according toclaim 1, wherein the composition further comprises serpentine.
 6. Amethod according to claim 1, wherein the irradiation is supplied by aWood's lamp which emits at about 375 nm.
 7. A method according to claim1, wherein the irradiation is supplied by a fluorescence microscopewhich emits at about 375 nm.
 8. A method according to claim 1, whereinthe composition is in the form of tablets or gelatine capsules.
 9. Amethod according to claim 8, wherein the composition contains 3 to 500mg of alstonine per unit dose, and further comprises a conventionalpharmaceutical support or carrier, and an excipient.
 10. A methodaccording to claim 1, wherein the tissue sample is treated in vivo in apatient, whereby the composition acts as a cytodiagnostic agent designedfor the selective detection of tumoral diseases.
 11. A method accordingto claim 10, wherein the composition is administered orally to thepatient.
 12. A method according to claim 1, wherein the tissue sample istreated in vitro.
 13. A method according to claim 12, wherein the tissuesample is treated by incubating in the composition for a period of 20 to40 minutes at ambient temperature.
 14. A method according to claim 13,wherein the tissue sample is incubated in an aqueous solution containingabout 200 μg of alstonine per ml.
 15. A method according to claim 14,wherein the irradiation is supplied by a Wood's lamp which emits atabout 375 nm.
 16. A kit for diagnosis of tumoral diseases and/or ofchromosomal aberrations in a tissue sample, comprising in packagedcombination(a) a composition comprising alstonine or an isomer thereofat a concentration effective to impart measurable fluorescence to tumorcells or to cells having chromosomal aberrations, and (b) an incubationsupport adapted for incubation of the tissue sample in the composition.17. A kit according to claim 16, further comprising a container of analcoholic wash solution.
 18. A kit according to claim 16, wherein thecomposition is a saturated aqueous solution of alstonine.
 19. An articleof manufacture comprising a packaging material and a cytodiagnosticagent contained within said packaging material, wherein saidcytodiagnostic agent comprises alstonine or an isomer thereof at aconcentration effective to impart a detectable level of fluorescence totumor cells, and wherein said packaging material comprises a label whichindicates that said cytodiagnostic agent can be used for the detectionof tumors and chromosomal aberrations.
 20. An article according to claim19, wherein the cytodiagnostic agent comprises alstonine or an isomerthereof at a concentration of from 1 to 25 weight % per volume.
 21. Anarticle according to claim 20, wherein the cytodiagnostic agent is agalenic preparation in the form of a tablet or capsule.